Clonal Dominance With Retroviral Vector Insertions Near the ANGPT1 and ANGPT2 Genes in a Human Xenotransplant Mouse Model

Abstract

Insertional leukemogenesis represents the major risk factor of hematopoietic stem cell (HSC) based gene therapy utilizing integrating viral vectors. To develop a pre-clinical model for the evaluation of vector-related genotoxicity directly in the relevant human target cells, cord blood CD34+ HSCs were transplanted into immunodeficient NOD.SCID.IL2rg−/− (NSG) mice after transduction with an LTR-driven gammaretroviral vector (GV). Furthermore, we specifically investigated the effect of prolonged in vitro culture in the presence of cytokines recently described to promote HSC expansion or maintenance. Clonality of human hematopoiesis in NSG mice was assessed by high throughput insertion site analyses and validated by insertion site-specific PCR depicting a GV typical integration profile with insertion sites resembling to 25% those of clinical studies. No overrepresentation of integrations in the vicinity of cancer-related genes was observed, however, several dominant clones were identified including two clones harboring integrations in the ANGPT1 and near the ANGPT2 genes associated with deregulated ANGPT1- and ANGPT2-mRNA levels. While these data underscore the potential value of the NSG model, our studies also identified short-comings such as overall low numbers of engrafted HSCs, limited in vivo observation time, and the challenges of in-depth insertion site analyses by low contribution of gene modified hematopoiesis.

Reference standards for accurate validation and optimization of assays that determine integrated lentiviral vector copy number in transduced cells

“…Assay 2 used a primer set and probe specific for a selected sequence within the gag’ portion in the 5′ sequence of integrated lentiviral genome. This assay was normalized via qPCR for a target sequence in the single copy PTBP2 (Polypyrimidine Tract Binding Protein 2) gene and the two reactions were performed in a single tube assay using optimized conditions for both gag and PTBP2 primer and probe sets (duplexed assay) 15 . Assay 3 targeted the RRE (Rev response element) sequence located downstream of gag’ in the integrated lentiviral genome.…”

Lost in translation: pluripotent stem cell‐derived hematopoiesis

“…Protocols for the expansion of HSPCs have therefore been adapted for the derivation of in vitro HSPCs from PSCs. Cytokines, such as SCF, TPO, FLT3‐L, IL‐6, and aryl hydrocarbon receptor signaling antagonizers (i.e., the small molecule StemRegenin), drive the expansion of cord blood CD34 + HSPCs while maintaining their CFU potential over a period of 10 days in culture (Haemmerle et al , ). The addition of IL‐3, however, favors the expansion and differentiation of CD34 + HSPCs on the expense of engraftment and reconstitution in vivo (Du et al , ).…”

Lentiviral Gene Therapy Using Cellular Promoters Cures Type 1 Gaucher Disease in Mice

“…In some cases, insertions with high read counts can indicate an increase in clonal abundance. 29,30 It is, however, not a direct measurement of clonal contribution, due to known biases in the PCR-based technology. 31 We further evaluated a possible overrepresentation of insertion sites in or near genes listed in cancer gene databases after in vivo selection ( Supplementary Table S3).…”